PROJECT SUMMARY Humans are frequently exposed to hazardous chemicals in the environment, which can damage DNA and lead to cancer. However, over the past 30 years, the measurements of DNA adducts have been largely carried out by monitoring a single or up to several DNA adducts, and the data have provided limited information about causative agents of cancer. There is a crucial unmet need to establish a robust technology to screen for a broad range of DNA adducts, some of which can be linked to mutations in cancer driver genes. The goal of our applica- tion is to advance our mass spectrometry (MS) adductomics methods to screen for an array of DNA adducts formed with hazardous chemicals in a single assay and develop algorithms for automated analysis. We will em- ploy high-resolution accurate mass spectrometry (HRAMS) to detect DNA adducts by extracting the charac- teristic spectral features of DNA adducts as they undergo fragmentation by the multistage (MSn) scanning. These MSn scanning approaches can detect DNA adducts of diverse structures in human tissues over a wide range of exposures. In this proposal, we will optimize our MS technology in rodents, under controlled exposures to an array of chemicals thought to contribute to renal cancer, and we will create a mass spectral database and develop software and informatics tools for comprehensive characterization of DNA adducts of the genome. In Aim 1, DNA adduct profiles will be generated in the kidney of rodents, following exposures to toxicants found in tobacco, the environment, diet, from pro-oxidants, and electrophiles produced endogenously. Panels of DNA adduct profiles resulting from these exposures will be characterized by untargeted data-dependent (DDA) and data-independent acquisition (DIA), employing nanoflow liquid chromatography and multistage MSn scanning with the Orbitrap MS. In Aim 2, a DNA adduct database will be constructed, and bioinformatics tools will be created for the unbiased and automated detection of DNA adducts by extraction of the mass spectral features of DNA adducts. The bioinformatics tools will identify patterns of adducts formed with different classes of renal toxicants and conduct systematic comparison and identification of DNA adducts based on different types of exposures and the mechanism of DNA damage. The DIA and DDA scanning methods, the DNA adduct database, and bioinformatics technologies will serve as templates to examine for different classes of DNA adducts in humans. In Aim 3, we will test our technologies to screen biopsies of renal cancer patients and explore tobacco exposure, one of the few recognized risk factors for this cancer. The proposed technology for molecular analysis of DNA adducts is amenable to other target organs and cell types and can help identify chemical agents and lifestyle factors that induce DNA damage and cancer risk. Identification of DNA-damaging agents can lead to strategies to mitigate or prevent exposures to hazardous chemicals with a dramatic impact on public health.